Volume 5, Issue 4, Pages 583-594 (October 2003) ER-to-Golgi Carriers Arise through Direct En Bloc Protrusion and Multistage Maturation of Specialized ER Exit Domains  Alexander A Mironov, Alexander A Mironov, Galina V Beznoussenko, Alvar Trucco, Pietro Lupetti, Jeffrey D Smith, Willie J.C Geerts, Abraham J Koster, Koert N.J Burger, Maryann E Martone, Thomas J Deerinck, Mark H Ellisman, Alberto Luini  Developmental Cell  Volume 5, Issue 4, Pages 583-594 (October 2003) DOI: 10.1016/S1534-5807(03)00294-6

Figure 1 Structure of PC-Containing Carriers HFs (A, D, and E) and CEFs (B and C) were subjected to hydroxylation blocks (HFs, 1% calf serum in the absence of ascorbic acid at 40°C for 3 hr; CEFs, 0.3 mM dipyridyl [see Bonfanti et al., 1998] for 1 hr). Cells were fixed at 0 (A and C–E), 4 (B), or 10 min (F–M) after hydroxylation block release (HFs, 32°C + 50 μM ascorbic acid; CEFs, after dipyridyl washout) and prepared for IF or IEM. Three-dimensional reconstruction and surface rendering of PC containers was performed on delineated serial sections. (A) ER-like pattern of the PC labeling before the release of the block (0 min). No colocalization of PC (green) is seen with Sec31 (red). PC colocalizes with calreticulin (not shown). (B) Four minutes after the release of the transport block, PC is in spots (green) that do not colocalize with Sec31 (red). (C) Diffuse distribution of PC (enhanced gold particles, arrow) through ER cisternae. (D) Distribution of Sec31 (enhanced gold particles, arrows) at an ERES and at ER cisternae (asterisks). (E) PC (DAB precipitate) is abundant in distended ER cisternae, whereas an ERES (arrows) is devoid of PC. (F) Ten minutes after the block release, folded PC (green) appears as spots offset from Sec31-positive spots (red). (G) PC (thin arrows) at the EM level is visible as gold aggregates in distended ER domains near ERESs (thick arrows). The PC staining does not have the diffuse ER appearance seen in (C). (H) The type II carrier. The PC container is connected (thick arrow) with the ER (arrowhead). Profiles of the ERES (thin arrow) do not contain PC. (I) Three-dimensional view of the type II carrier shown in (C). The PC-positive container is in red, and the ER is in white. The PC-negative container is in yellow. (J and K) Type III carrier. Serial sections of the tangential tubule (thin arrow) containing a varicosity with PC (DAB precipitate; thick arrow). (L) Type IV carrier. Two saccules are filled with PC (thick arrows), whereas profiles of an ERES (thin arrow) do not contain PC. (M) Three-dimensional view of the type IV carrier shown in (H). The ERES is in yellow, the ER in green, and the PC-positive container is in red. The scale bars represent 5 μm (A), 2 μm (B and F), 100 nm (C–E and L), 200 nm (G and H), and 350 nm (J and K). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 2 Initial Stages of VSVG Exit from the ER HFs (A–D) and COS7 (E–K) cells were infected with 045VSV, placed at 40°C for 3 hr to accumulate VSVG in the ER (HFs were grown in the presence of 10% FCS to inhibit the synthesis of PC), shifted back to 32°C, fixed 0 (A–D) or 4 (E–K) min after the release of the temperature block, and prepared for IF or IEM. (A–C) Reticular pattern of VSVG ([B and C], red) and no colocalization with Sec31 ([A and C], green). Sec 31 reveals a significant background. (D) Sec31 labeling of ER cisternae (asterisks) adjacent to an ERES (arrow). (E and F) Emanation of tubules positive for VSVG (E) and Sec31 (F) from the ER 4 min after the release of the temperature block. (G) Three-dimensional representation of COPI localization by immunoperoxidase, in the presence of 12% gelatin to prevent diffusion of the DAB precipitate. Analogous results were obtained by nanogold labeling. (H–K) Concentration of total VSVG ([I]; red, detection with anti-cytosolic domain antibodies), folded VSVG ([K]; blue, detection with I-14 antibodies; Lefrancois and Lyles, 1983), and ERESs ([J]; green, labeled with anti-Sec23). Both unfolded and folded VSVG are concentrated at ERESs. Diffuse VSVG staining is barely detectable only for the unfolded form. (L) Colocalization of PC, VSVG, COPII (Sec23), and COPI at various times after release of the exit block. The quantification is from confocal sections, according to Mironov et al. (2001). Bars are standard errors from the quantification of 20 cells in each case. (M) VSVG concentration (linear density) at ER exit domains at various times after release of the exit block. Exit domains include both the ERES and the forming saccular carrier and are defined as described by Klumperman et al. (1998). The quantification is from cryosections labeled with immunogold and from epon sections labeled by nanogold (then gold enhanced) at the preembedding step. The linear density of gold particles at exit domains was normalized to the linear density of gold particles on ER membranes. Bars are standard errors from the quantification of 20 cells in each case. At times 0 and 20 min, the quantification was performed only on cryosections. The scale bars represent 7.5 μm (A–C), 240 nm (D), 300 nm (E and F), and 2.5 μm (G–J). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 3 Structure of VSVG-Containing Carriers COS7 (A, B, and I–L) and RBL (C–H) cells were treated as described in the legend to Figure 2, fixed 10 min after the release of the ER exit block, and prepared for IF or for peroxidase IEM. After IEM labeling, serial sections were prepared and 3D reconstruction and surface rendering were performed. (A) Concentration of folded VSVG in the peripheral spots (red) is offset from Sec31-positive spots (green). The arrow shows the VSVG-positive tubule. (B) A type I carrier (white asterisk) widely connected (arrows) to the ER (black asterisks) in the vicinity of an ERES (arrowheads). (C) Representative serial section of a type II carrier. The arrow shows the EGC positive for VSVG. (D) Surface rendering (derived from [G]) of a type IV carrier (red) in close vicinity to an ERES (yellow) and the ER (white). (E and F) Surface rendering (derived from [C]) of a type II carrier (red) in close vicinity to an ERES (yellow in [E], omitted in [F]) and the ER (white). (G) Type IV carrier with three saccular domains filled with VSVG (DAB precipitate) in close vicinity to an ERES (arrow) and the ER (asterisks). (H) Type III carriers appearing on a 200 nm tangential section as varicosities (arrows) along a thin radial tubule. (I and J) Thick (250 nm) sections of carriers labeled for VSVG (DAB precipitate) were cut, prepared for electron tomography, and virtual 2–3 nm slices (e.g., in [I]) were extracted from the tomograms. Three-dimensional reconstruction and surface rendering of the VSVG-containing carrier (red) and the ER (yellow) were performed (see [J]). The arrows in [I] and [J] show the sites of connection between the ER and the VSVG container. (K) Serial ultrathin (30 nm) sections of a carrier from rapid freezing-cryosubstitution were cut and used to form a 3D reconstruction of the image (see [L]). The black arrows indicate the ER and its connection to the ERES (white arrow). (L) Saccular container (red) with a blebby surface connected to the ER (green). Six containers were examined by this approach, and they were all connected to the ER. The scale bars represent 1.5 μm (A), 150 nm (B, C, I, and K), 200 nm (G), and 150 nm (H). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 4 The Carrier Life Cycle by CVEM and CLEM Analyses COS7 cells were transfected with VSVG-FP, placed in glass-bottomed microwell dishes with coordinated grids, kept for 12 hr at 40°C to accumulate VSVG-FP in the ER, shifted to 32°C to release VSVG from the ER, and then examined by time-lapse analysis under the laser-scanning confocal microscope (A) and prepared for CVEM (B–J). COS7 cells were also grown on dishes with coordinated grids (K–M), treated as described in the legend to Figure 3, fixed, and prepared for CLEM. Next, VSVG, COPII, and COPI were visualized with IF, and COPII-, COPII/COPI-, and COPI-positive carriers were selected after Z stacking under the laser-scanning confocal microscope, and cells were embedded in epon and serially cut, with their subsequent identification at the EM level. (A) Inverted video frames showing the emanation of thin VSVG-FP-positive tubules from the VSVG-FP-positive spots on their way toward the Golgi. (B and C) Representative serial sections of type I carriers, which were subjected to 3D reconstruction ([I] and [H], respectively) after their analysis by videomicroscopy. Six other carriers examined at a similar stage in their life cycle all showed a similar ultrastructure. (D–G) Representative serial sections of a slowly moving, type IV carrier (D); a type II carrier just before its centripetal movement ([E]; three such carriers were examined); and two quickly moving, type III carriers detected under the nucleus (F and G). At the IF level, the last of these (type III) appear as bright varicosities along the less bright fluorescent tubule. Two other carriers of type III exhibiting the same type of mobility were examined. (H–J) Three-dimensional reconstruction and surface rendering of ER-to-Golgi containers ([H], from [C]; [I], from [B]; [J], from [E]) (red) and ER in white (H and I) or green (J). An ERES is in yellow (J). (K–M) Examples of COPII-positive ([K], type I), COPII/COPI-positive ([L], type II), and COPI-positive ([M], type IV) carriers identified by CLEM. The scale bars represent 4 μm (A), 140 nm (B, D–F, and K–M), and 100 nm (C and G). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 5 PC and VSVG Are Concentrated in Different Domains of the ER but Move to the Golgi by the Same Transport System HFs were stimulated to synthesize PC by adding 1% calf serum, then infected with 045VSV and incubated at 40°C for 3 hr in the absence of ascorbic acid to accumulate both PC and VSVG in the ER. Next, cells were shifted to 32°C and 50 μg/ml of ascorbic acid was added back to the medium to release both the temperature and hydroxylation blocks. The HFs were fixed at 0 (not shown), 4 (A–D and I–K), or 10 (E–H, L, and M) min after the temperature shift. Cells were prepared for IF or IEM. (A–D) Initial concentration (4 min) of folded VSVG (red) and folded PC (blue) in different ER domains adjacent to an ERES (Sec23, green). (E–H) Coalescence (10 min) of folded VSVG (red) and folded PC (blue) in the carriers in the vicinity of an ERES (Sec23, green). (I) Colocalization of VSVG (10 nm particles) and COPII (15 nm particles) in the same domains of the ER (presumably exit domains) 4 min after release of the block. (J) Lack of precise colocalization between PC (15 nm particles) and COPII (10 nm particles) 4 min after the release of the block. (K) Concentration of VSVG (10 nm particles) and PC (15 nm particles) in different domains of the ERES 4 min after the release of the block. (L) Colocalization of VSVG (10 nm particles) and PC (15 nm particles) in the same carriers in the vicinity of the Golgi 10 min after release of the block. (M) Colocalization of VSVG (10 nm particles) and COPI (15 nm particles) in the same carriers 10 min after the release of the block. er, endoplasmic reticulum; g, Golgi complex; m, mitochondria; n, nucleus. The scale bars represent 0.3 μm (A–D and E–H), 150 nm (I, K, and M), and 100 nm (J and L). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 6 VSVG Exit Does Not Involve 60 nm Vesicles In Vivo but Depends on the COPII Machinery RBL (A) and COS7 (C–F) cells were treated as described in the legend to Figure 3, or COS7 cells (B) were infected and kept at 32°C for 2 hr. The cells were chemically fixed and prepared for IEM (A–C), or transfected with ssHRP, fixed 24 hr after transfection (at steady state), and then processed for detection of ssHRP (Connolly et al., 1994; D–F). (A) Permeabilization of cells with 0.1% Triton X-100 neither changes the structure of the VSVG containers (thin arrow) or the ER (thick arrows), nor induces the appearance of labeling in round profiles (arrowheads). (B and C) VSVG (visualized with the P5D4 monoclonal antibodies against the VSVG cytosolic domain) is concentrated in a saccular domain (arrowhead), which is connected to the ER (thick arrow in [C]), but is not present in round profiles of ERESs (thin arrows). The structure of the VSVG containers (in [B]) at steady state is similar to that after cargo synchronization. (D–F) ssHRP is present in saccular carriers (arrowheads) but not in profiles of ERESs (arrow in [D]). (G–J) VSVG (red) is concentrated in the peripheral Sec31-positive (green) spots in NSF antibody-microinjected cells (detected by Cy5; blue in [G]). Microinjection with anti-NSF and anti-p97 antibodies. BHK cells (G–N) were infected with 045VSV, and after accumulation of VSVG in the ER (see legend to Figure 2) at 40°C, they were microinjected (at 40°C) with the inhibitory anti-NSF (G–J and K–M) or anti-p97 (N) antibodies mixed with anti-mouse Fab fragments conjugated with Cy5 (see Experimental Procedures). After an additional 30 min incubation at 40°C, the cells were shifted to 32°C and fixed after 10 min. Injected cells were found using CLEM (not shown). In mockinjected cells (arrow in [I]), VSVG was also found in the Golgi area. Cells were prepared for IF or IEM (nanogold-enhanced, [K]; immunoperoxidase, [L–N]). (K–N) Generation of type II (K, L, and N) and type IV (M) carriers. VSVG exiting from the ER concentrates in saccular domains (arrows) of the ERESs. No accumulation of VSVGcontaining round profiles (arrowheads) was seen. (O) Microinjection of wild-type Sar1p does not interfere with the exit of PC (red) from the ER. The inset shows the FITC-dextran that was injected together with Sar1p. (P and Q) Microinjection of Sar1p-GDP inhibits the exit of PC (red) from the ER. The inset shows the FITC-dextran that was injected together with Sar1p-GDP. The arrow shows a noninjected cell. Microinjections with GDP-restricted Sar1p. HFs cells were microinjected with tagged versions (see Experimental Procedures) of either wild-type Sar1p or GDP-restricted Sar1p at 40°C. Thirty minutes after microinjection, the exit block was removed and the cells were examined by immunofluorescence 10 min after the shift. A similar trial was performed on VSVG exit from the ER, with similar results. The scale bars represent 100 nm (A), 140 nm (B and C), 120 nm (D), 60 nm (E and F), 5 μm (G–J and O), 80 nm (K–N), and 10 μm (P and Q). Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)

Figure 7 Schematic Representation of ER-to-Golgi Carrier Types Procollagen is drawn in brown thin lines; VSVG, in green dots; COPII, in blue dashed line; and COPI, in red spheres. (A and B) Type I. Distended domains of the ER with concentrated cargo are located close (from a few nm to 0.5 μm) to an ERES and are likely to be containers fixed just in the process of appearing from the ER. ERESs (A and B) and VSVG carriers (B) are coated with COPII, while PC carriers (A) are not. PC is excluded from ERESs, whereas VSVG often partially penetrates them. (C and D) Type II. Flattened and elongated (>300 nm) saccules protruding from, but still in continuity through tubules with, the ER. These develop from type I carriers after protrusion from the ER. COPI is localized at the isthmus of the protruded carrier (D). (E and F) Type III. Distensions (>300 nm in length) embedded in thin (50–70 nm) tubules devoid of ribosomes, which are usually radially oriented. They appear to be carriers caught during translocation toward the Golgi. (G and H) Type IV. Larger and more complicated membranes comprising several (two to four) saccules partially stacked and associated with the ER. The association with the ER is represented with a dashed line. (I) Type I carriers containing both VSVG and PC in the same cell (HF). PC and VSVG do not share the same zone of the ER before exit. (J) Development of the carriers as inferred from CVEM experiments. Developmental Cell 2003 5, 583-594DOI: (10.1016/S1534-5807(03)00294-6)